Polypeptides and peptides have become increasingly important agents in a variety of applications, including industrial applications and use as medical, therapeutic and diagnostic agents. However, in certain physiological states, such as Cancer and inflammatory states (e.g., COPD), the amount of proteases present in a tissue, organ or animal (e.g., in the lung, in or adjacent to a tumor) can increase. This increase in proteases can result in accelerated degradation and inactivation of endogenous proteins and of therapeutic peptides, polypeptides and proteins that are administered to treat disease. Accordingly, some agents that have potential for in vivo use (e.g., use in treating, diagnosing or preventing disease) have only limited efficacy because they are rapidly degraded and inactivated by proteases.
Protease resistant polypeptides provide several advantages. For example, protease resistant polypeptides remaining active in vivo longer than protease sensitive agents and, accordingly, remaining functional for a period of time that is sufficient to produce biological effects. A need also exists for improved methods to select polypeptides that are resistant to protease degradation and also have desirable biological activity.
VEGF:
VEGF is a secreted, heparin-binding, homodimeric glycoprotein existing in several alternate forms due to alternative splicing of its primary transcript (Leung et al., 1989, Science 246: 1306). VEGF is also known as vascular permeability factor (VPF) due to its ability to induce vascular leakage, a process important in inflammation.
An important pathophysiological process that facilitates tumor formation, metastasis and recurrence is tumor angiogenesis. This process is mediated by the elaboration of angiogenic factors expressed by the tumor, such as VEGF, which induce the formation of blood vessels that deliver nutrients to the tumor. Accordingly, an approach to treating certain cancers is to inhibit tumor angiogenesis mediated by VEGF, thereby starving the tumor. Avastin (bevacizumab; Genetech, Inc.) is a humanized antibody that binds human VEGF that has been approved for treating colorectal cancer. An antibody referred to as antibody 2C3 (ATCC Accession No. PTA 1595) is reported to bind VEGF and inhibit binding of VEGF to epidermal growth factor receptor 2.
Targeting VEGF with currently available therapeutics is not effective in all patients, or for all cancers. Thus, a need exists for improved agents for treating cancer and other pathological conditions nediated by VEGF e.g. vascular proliferative diseases (e.g. Age related macular degeneration (AMD)).
VEGF has also been implicated in inflammatory disorders and autoimmune diseases. For example, the identification of VEGF in synovial tissues of RA patients highlighted the potential role of VEGF in the pathology of RA (Fava et al., 1994, J. Exp. Med. 180:341:346; Nagashima et al., 1995, J. Rheumatol. 22: 1624-1630). A role for VEGF in the pathology of RA was solidified following studies in which anti-VEGF antibodies were administered in the murine collagen-induced arthritis (CIA) model. In these studies, VEGF expression in the joints increased upon induction of the disease, and the administration of anti-VEGF antisera blocked the development of arthritic disease and ameliorated established disease (Sone et al., 2001, Biochem. Biophys. Res. Comm. 281: 562-568; Lu et al., 2000, J. Immunol. 164: 5922-5927). Hence targeting VEGF may also be of benefit in treating RA, and other conditions e.g. those associated with inflammation and/or autoimmune disease.